The earliest of T cells exist mainly in bone marrow or fetal liver, as a form of lymphoid stem cells differentiated from hematopoietic stem cells having an ability to differentiate into multilineage cells. These earliest stem cells emigrate to the thymus and mature into T cells through a series of differentiation stages. The differentiation and maturation of the T precursor cells are greatly under the influence of thymic stromal cells. After T precursor cells enter into the thymic cortex, the T precursor cells differentiate and mature into T cells through rapid cell division and proliferation under the control of adhesion with stromal cells comprising mainly thymic epithelial cells which participate in differentiation and maturation of the T precursor cells, adhesion with myeloid cells such as macrophages, and humoral factors produced therefrom. After undergoing several differentiation stages thymocytes flow out to the bloodstream from the cortico-medullary junction of thymus, and emigrate to peripheral lymphoid organs. The emigrated thymocytes, i.e., T cells, further differentiate and mature therein.
To study the detailed process of differentiation and maturation of the T precursor cells, a culture system capable of achieving in-vitro proliferation, differentiation, and maturation of T cells is required.
On the other hand, it is considered that abnormality of differentiation or of functions of T cells due to immunization with infectious bacteria and/or an foreign antigen is one great cause of allergic diseases or various autoimmune diseases. (e.g., Animesh A. et al, Science, Vol. 248, 1380-1388, 1990; Irun R. Cohen et al, Immunology Today, Vol. 12, No. 4, 105-110, 1991; and Lars Klareskog et al, Immunological Reviews, Vol. 118, 285-310, 1990).
At present, as therapeutic drugs for such diseases, non-specific anti-inflammatory agents and immunosuppressants of T cell functions are used as suppressors. However, essential cure of the diseases has not been achieved due to their non-specificity.
To overcome these problems, extensive studies on vaccine therapy and serum vaccine therapy have been made, comprising respectively (1) specifying an antigen which causes disease and applying the antigen, and (2) specifying said antigen and applying suppressive factors specific for the diseases which are represented by antibodies against the antigen. A cell vaccine therapy using T cells whose antigen specificities has been proved is considered to be an effective therapeutic method for diseases which is assumed to be caused by abnormal differentiation and functional abnormality of T cells (e.g., Jose C. Gutierrez-Ramos et al, Nature, Vol. 346, 271-274, 1990 and Kohei Ota et al, Nature, Vol. 346, 183-187, 1990). However, methods capable of deriving T cells having appropriate antigen specificity in vitro are limited to a particular method.
In fact, although a submerged (suspension) culture in which cells or tissues are submerged or suspended in a nutrient medium is well known as a method generally used for culture of cells or tissues, it is impossible to differentiate and mature T precursor cells into T cells in accordance with such a conventional method.
At present, as the sole culture method capable of achieving differentiation and maturation of the T precursor cells, only a so-called afloat culture is known, in which a fetal thymus is cultured on an interface between the gas phase and the nutrient medium under atmospheric air whose carbon dioxide concentration is controlled to 5% (e.g., Jenkinsen E. J. et al, Eur. J. Immunology, Vol. 12, 583, 1982 and Osamu Mazda et al, J. Exp. Med., Vol. 173, 539-547, 1991). However, this method requires special tools or apparatuses, and differentiation of cells on the liquid surface side is insufficient. In addition to these drawbacks, this method is not suitable for the culture of the T precursor cells accompanying mass proliferation of cells. Therefore, it is difficult to perform a large scale culture.
Strong demand has, therefore, arisen for developing a culture method capable of realizing and achieving derivation of matured T cells differentiated from T precursor cells in terms of their phenotypes of differentiation antigens and of their functions, by using the submerged (suspension) culture as a general method of culturing cells or tissues.
Japanese Unexamined PCT Application No. 3-504329 discloses a method for proliferating a human B blastic cell line by raising the dissolved oxygen concentration in the culture medium by steadily supplying a desired amount of oxygen into the medium placed in a sealed vessel, through a silicon pipe connected to a gas bomb sealed with a gas mixture containing a desired amount of oxygen. In said culture according to the method, the proliferation of the human B blastic cell line is found but differentiation and maturation of the cells is not achieved. The present invention is capable of achieving proliferation of T precursor cells, and of differentiation and maturation of them in terms of their phenotypes of differentiation antigens and of their functions, by using a simpler culture method than that disclosed in above application under a higher concentration of dissolved oxygen in the medium.